ANTIMICROBIAL ANGIOGENIN COMPLEXES (ANGex) AND USES THEREOF

ABSTRACT

Antimicrobial compositions based upon stabilized angiogenin compositions also contain osteopontin and antimicrobial proteins such as lactoperoxidase (LPO), myeloperoxidase (MPO), salivary peroxidase (SPO) and lysozyme.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/185,115, filed Jul. 18, 2011, which is a divisional of U.S.application Ser. No. 12/554,602, filed Sep. 4, 2009, now U.S. Pat. No.8,003,603, issued Aug. 23, 2011, which is a divisional of U.S.application Ser. No. 11/734,729, filed Apr. 12, 2007, now U.S. Pat. No.7,601,689 issued Oct. 13, 2009. All of the above listed applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to protein stabilization, particularlystabilization of angiogenin by immobilization on natural substrateswhich includes but not limited to proteins, polysaccharides, lipids andpolyphenols.

2. Description of the Related Art

Angiogenesis and vasculogenesis are two primary pathways in thedevelopment and maintenance of mammalian health. The angiogenic role isto supply and support tissue with ample vasculature, thus providing aroute of access for the transportation of essential nutrients, includingoxygen and the removal of metabolic waste in a sustained manner.Angiogenesis is a strictly regulated, multi-step process that occursduring normal physiology such as wound healing, pregnancy, anddevelopment.

Angiogenin (ANG) has been shown to be a key mediating factor in theunderlying cascade of chemical events leading to angiogenesis, whichmakes it a very important precursor molecule for both muscle developmentand vascular generation. ANG is a 14-kDa, basic heparin-binding proteinand a member of the pancreatic ribonuclease (RNase) superfamily. ANG canserve as a substrate for endothelial cell adhesion. ANG resemblespancreatic RNase-A; their amino acid sequences are about 35% identical,including the active site residues. An overview of the relationship ofANG and other RNases of the super-family showed that their genes all arein relative close proximity on human chromosome 14. However, human ANGshows a weak ribonucleolytic activity (lower by 10⁴ to 10⁶-fold) despiteof its potent angiogenic function. The actions of ANG involve nearly allphases of angiogenesis (Strydom D J. Cell Mol. Life. Sci. 54:811-824,1998; Acharya B et al., Proc. Natl. Acad. Sci. USA 91:2915-2919, 1994).When ANG was implanted into experimentally injured menisci of NewZealand white rabbits, localized neovascularization occurred in 52% ofthe treated animals as compared to 9% of controls (King T V et al., J.Bone Joint Surg. Br. 73(4):587-590, 1991). Mutant ANG proteins withenhanced angiogenic activity have also been reported (WO 89/09277). Sitespecific mutations in ANG resulted in mutant proteins with increasedRNase and angiogenic activities (U.S. Pat. No. 4,900,673). Replacementof a specific section of ANG with a subsequence characteristic of RNaseunexpectedly resulted in a mutant ANG/RNase hybrid with increasedangiogenic activity (U.S. Pat. No. 5,286,487; U.S. Pat. No. 5,270,204).

ANG (RNase type-4 and RNase type-5 forms) is an active secretory proteinfound in milk. In cow's milk the concentrations are about 2 mg/L forRNase 4 and between 1 and 8 mg/L for RNase 5 (Ye X Y, et al., Life Sci.67:2025-2032, 2000; Komolova G S, et al., Appl. Biochem. Microbiol.38:199-204, 2002). ANG circulates in human plasma at a concentration ofabout 0.3 μg/mL with a fast turnover rate and a half-life <5 min. ANGcan induce most of the events necessary for the formation of new bloodvessels. It binds avidly to endothelial cells and stimulates cellmigration and invasion. ANG promotes cell proliferation anddifferentiation; mediates cell adhesion and activates cell associatedproteases; and also induces plasminogen activator and thereby, theplasmin system promoting migration and tubular morphogenesis ofendothelial cells. Exogenous ANG is transported into the nucleus ofendothelial cells. The nuclear translocation results in accumulation ofthe ANG in the nucleolus. Transportation of ANG from the cell surfaceinto the nucleus and subsequently to the nucleolus is critical for itsangiogenic activity. The import of ANG from the cytosol to the nucleusis signal-dependent, carrier mediated and energy-dependent, activetransport process (Hu G F, et al., Proc. Natl. Acad. Sci. USA94:2204-2209, 1997; Moroianu J, et al., Proc. Natl. Acad. Sci. USA91:1677-1681, 1994).

ANG is a potent inducer of neo-vascularization and the only angiogenicmolecule known to exhibit ribonucleolytic activity. Its overallstructure, as determined at 2.4 Å, is similar to that of pancreaticRNase A, but it differs markedly in several distinct areas, particularlythe ribonucleolytic active center and the putative receptor bindingsite, both of which are critically involved in biological function. Moststrikingly, the site that is spatially analogous to that for pyrimidinebinding in RNase A differs significantly in conformation and is“obstructed” by Gln-117. Movement of this and adjacent residues may berequired for substrate binding to ANG and, hence, constitute a key partof its mechanism of action (Acharya K R, et al., Proc. Natl. Acad. Sci.USA 91:2915-2919, 1994; Russo N, et al., Proc. Natl. Acad. Sci. USA91:2920-2924, 1994).

X-ray diffraction and mutagenesis results have shown that the activesite of the human protein is obstructed by Gln-117 and imply that theC-terminal region of ANG must undergo a conformational rearrangement toallow substrate binding and catalysis. Two residues of this region,Ile-119 and Phe-120, make hydrophobic interactions with the remainder ofthe protein and thereby help to keep Gln-117 in its obstructiveposition. Furthermore, the suppression of activity by theintra-molecular interactions of Ile-119 and Phe-120 is counter-balancedby an effect of the adjacent residues, Arg-121, Arg-122 and Pro-123,which do not appear to form contacts with the rest of the proteinstructure. They contribute to enzymatic activity by constituting aperipheral sub-site for binding polymeric substrates. These resultsreveal the nature of the conformational change in human ANG and assign akey role to the C-terminal region both in this process and in theregulation of human ANG function (Russo N, et al., Proc. Natl. Acad.Sci. USA 93:3243-3247, 1996).

The pioneering work of Vallee and co-workers has paved the path in thedevelopment of health applications for human ANG. U.S. Pat. No.4,727,137 discloses therapeutic use of human ANG to promote thedevelopment of hemo-vascular network, for example, to induce collateralcirculation following a heart attack, or to promote wound healing, forexample, in joints or other locations. This invention also describesdiagnostic applications of human ANG in screening for malignancies. U.S.Pat. No. 4,952,404 describes healing of injured avascular tissue couldbe promoted by applying human ANG in proximity to the injured tissue.

Besides an angiogenic factor, ANG has been used in the treatment ofviral infection such as HIV (WO 2004/106491A2). The RNase activity ofANG seem to be an inhibitor of viral replication.

Activation of the receptor for ANG has been proposed as a method topromote wound healing (WO 98/40487A1). A method of skin whitening byapplying a composition containing ANG has been described (U.S. Pat. No.5,698,185). ANG was first isolated from human carcinoma cells andsubsequently from human plasma, bovine plasma, bovine milk, mouse,rabbit, and pig sera and goat plasma (Maiti T K, et al., Prot. Pep.Lett. 9:283-288, 2002) and its use to diagnose cancer has been suggested(WO 02/25286).

However, the exploitation of human ANG polypeptide for broad-spectrumhuman health-care (e.g., health supplementation, body building,cosmetics, oral health, post-operative wound care) and animal healthapplications (e.g., feed conversion for weight gains in meat-yieldinganimals) is limited without a mass supply of the compound. Such massproduction of ANG requires an acceptable (preferably a food-grade) rawmaterial source and an effective large-scale purification process for ahigh yield of ANG. Isolation of milk ANG from healthy dairy animalscould provide an answer to this limitation.

Bovine Milk ANG

Spik and co-workers described a method to isolate ANG from mammalianmilk. U.S. Pat. No. 5,171,845 discloses an extraction process for ANGfrom cow milk consisting of a delipidation step by centrifugation,chromatographic steps on SP-Sephadex® C50 and S-Sepharose® columns,followed by a gel filtration step on Bio-gel® P-30 column with a finalfast protein liquid chromatography (FPLC) step on Phenyl Superose® HR5/5column. The protein yield was estimated at 0.5 mg of ANG per liter ofdelipidated milk.

U.S. Pat. Nos. 6,010,698 and 6,268,487 disclose alternative processesfor isolating ANG (their homologues and fragments) from mammalian milkor a milk derivative.

Bovine milk ANG is a single-chain protein of 125 amino acids; itcontains six cysteines and has a calculated molecular weight of 14,595.Bovine milk ANG has 65% sequence homology with human plasma ANG and 34%homology with bovine pancreatic RNase A. The three major active siteresidues involved in the catalytic process, His-14, Lys-41 and His-115,are conserved in the bovine milk ANG with ribonucleolytic activitycomparable to that of the human protein. Bovine milk ANG contains anadditional cell recognition tri-peptide Arg-Gly-Asp, which is notpresent in the human ANG protein. In contrast to the human protein, theN-terminus of bovine ANG is unblocked. Two regions, 6-22 and 65-75, arehighly conserved between human and bovine ANG proteins, but aresignificantly different from those of the RNases, suggesting a possiblerole in the molecules' biological activity. Bovine ANG has the followingsequence:NH₂-Ala(1)-Gln-Asp-Asp-Tyr-Arg-Tyr-Ile-His-Phe(10)-Leu-Thr-Gln-His-Tyr-Asp-Ala-Lys-Pro-Lys(20)-Gly-Arg-Asn-Asp-Glu-Tyr-Cys-Phe-Asn-Met(30)-Met-Lys-Asn-Arg-Arg-Leu-Thr-Arg-Pro-Cys(40)-Lys-Asp-Arg-Asn-Thr-Phe-Ile-His-Gly-Asn(50)-Lys-Asn-Asp-Ile-Lys-Ala-Ile-Cys-Glu-Asp(60)-Arg-Asn-Gly-Gln-Pro-Tyr-Arg-Gly-Asp-Leu(70)-Arg-Ile-Ser-Lys-Ser-Glu-Phe-Gln-Ile-Thr(80)-Ile-Cys-Lys-His-Lys-Gly-Ser-Ser-Arg(90)-Pro-Pro-Cys-Arg-Tyr-Gly-Ala-Thr-Glu-Asp(100)-Ser-Arg-Val-Ile-Val-Val-Gly-Cys-Glu-Asn(110)-Gly-Leu-Pro-Val-His-Phe-Asp-Glu-Ser-Phe(120)-Ile-Thr-Pro-Arg-His-COOH(SEQ ID NO: 1). Disulfide bonds link Cys(27)-Cys(82), Cys(40)-Cys(93),and Cys(58)-Cys(108) (Maes P, et al., FEBS Lett. 241:41-45, 1988; Bond MD, et al., Biochemistry 28:6110-6113, 1989).

Molecular dynamics simulation (MDS) studies showed marked differences inthe hydrogen-bonding patterns in the active site regions of the humanand bovine ANG systems. Furthermore, the positions of water moleculesidentified in the crystal structures of human ANG significantly differfrom that of the bovine ANG. Positioning of the water molecules in theprotein structure play an important role in manifesting the subtlefunctional differences between human and bovine ANG systems (MadhusudhanM S, et al., Biopolymers 49:131-144, 1999).

Synthetic peptides corresponding to the C-terminal region of ANG inhibitthe enzymatic and biological activities of the molecule, while peptidesfrom the N-terminal region do not affect either activity. SeveralC-terminal peptides also inhibit the nuclease activity of ANG when tRNAis the substrate. Furthermore, peptide Ang(108-123) decreases theneo-vascularization elicited by ANG in the chick chorioallantoicmembrane assay (Ryback S M, et al., Biochem. Biophys. Res. Commun.162:535-543, 1989).

The mechanism of the angiogenic activity involves multiple interactionsof ANG with various molecules through specific regions on its proteinsurface. The interactive molecules include heparin, plasminogen,elastase, angiostatin, actin and most importantly a 170-kilodaltonreceptor on sub-confluent endothelial cells.

The interaction of ANG with heparin could protect the molecule fromprotein cleavage by trypsin hydrolysis. A basic ‘triple’ amino acidcluster on ANG, Arg-31/Arg-32/Arg-33, has been identified as the heparinbinding site. Mutations of the triple cluster and of the Arg-70 residuecould decrease the binding affinity of ANG to heparin as well as itscell adhesion property. However, a replacement of any other basicresidues in the polypeptide chain does not affect the heparin bindingproperty of ANG. The heparin binding site on ANG is outside thecatalytic center. Light scattering measurements on ANG-heparin mixturessuggest that a single heparin chain (mass of 16.5 kDa) could interactwith approximately 9 ANG molecules (Soncin F, et al., J. Biol. Chem.272:9818-9824, 1997).

Several bio-molecules in milk and other exocrine secretions avidly bindto heparan sulfate, the active constituent of “mucin” that overlay theintestinal epithelia. The heparan sulfate interaction is generallymediated by cationic domains located in the N-terminus region of suchbio-molecules. These immobilization processes facilitate retention ofbiological compounds on epithelial surface and could possibly “activate”these molecules for specific physiological functions, including theirinternalization and bioavailability. Accordingly, heparan sulfate andits analogues have a widespread application in chromatography as columnmatrices, for purification and isolation of several milk compounds,including ANG, lactoferrin, lactoperoxidase and other bioactivepeptides.

U.S. Pat. No. 6,172,040 describes a method for immobilization of milklactoferrin (LF) on a galactose-rich polysaccharide (GRP) substrate,which is analogous to the heparan sulfate. This immobilization processinvolved the interaction of GRP with a highly cationic N-terminus domainof LF, as expected. The immobilization process described in thisinvention caused a significant increase in the antimicrobial activity ofLF, and also provided a structure-conformational stability to theprotein molecule.

The binding of ANG to heparan sulfate via its cationic N-terminusdomain, its mitogenic characteristics and occurrence in differentphysiological milieu such as milk, plasma, other exocrine secretions andtissue sites, is in striking proximity to LF. On a speculative basis,the immobilization methods for LF, which are disclosed in U.S. Pat. No.6,172,040, when adapted and applied to ANG, this angiogenic milk proteinhas demonstrated a unique molecular and functional behavior.

SUMMARY OF THE INVENTION

The present invention relates to immobilized ANG complexes (ANGex) andmixtures of ANGex and free dispersed native (fdn)-ANG having preserved,enhanced and specific biological activity. ANG is immobilized onnaturally occurring substrates, preferably via the N-terminus region ofthe ANG. Suitable substrates include proteins, polysaccharides,cellulose compounds, nucleic acids, nucleotides, lipids and metalliccompounds. Preferred substrates include collagen, gelatin, fibronectin,casein, mucin, heparin-sulfate, carrageenan, gums/galactans, pectins,deoxyribonucleic acid, adenosine triphosphate or a triglyceride,galactose-rich polysaccharide (GRP), vitamin-E, ceruloplasmin,metallo-thionein and lactoferrin (LF) being most preferred.

Embodiments of the invention are directed to compositions which includean isolated angiogenin non-covalently complexed to an isolated naturallyoccurring substrate.

In preferred embodiments, the angiogenin is isolated from a biologicalfluid. Preferably, the biological fluid is colostrum, milk, whey, milkserum, blood, plasma or serum. Preferably, the biological fluid isobtained from a mammal which is selected from humans, cows, buffalos,horses, sheep, pigs and camels. In some embodiments, the mammal isgenetically modified.

In preferred embodiments, the substrate includes proteins such astransport proteins, subepithelial matrix proteins, or antimicrobialproteins. Preferably, the transport protein is lactoferrin, transferrin,ovo-transferrin (conalbumin), ceruloplasmin or transfer factors.Preferably, the subepithelial matrix protein is fibronectin, fibrinogen,laminin, vitronectin, osteopontin, native collagens or denaturedcollagen (gelatin). Preferably, the antimicrobial protein is selectedfrom peroxidases (lacto, myelo and salivary forms) and lysozyme.

In some preferred embodiments, the substrate includes triglycerides.

In some preferred embodiments, the substrate includes a coenzyme such ascoenzyme-Q10 (ubiquinone) or NADH.

In some embodiments, the substrate includes a vitamin such as vitaminsA, C and/or E.

In some embodiments, the substrate includes a nucleic acid such assingle and double stranded DNA and RNA. In some embodiments, thesubstrate includes a nucleotide such as ATP, CTP, GTP or TTP.

Embodiments of the invention are directed to methods of preparingangiogenin complexes by mixing the angiogenin with a substrate in aliquid medium. In preferred embodiments, the substrate includes aprotein such as transport proteins, subepithelial matrix proteins andantimicrobial proteins. Preferably, the transport protein includeslactoferrin, transferrin, ovo-transferrin (conalbumin), ceruloplasminand/or transfer factors. Preferably, the subepithelial matrix proteinincludes fibronectin, fibrinogen, laminin, vitronectin, osteopontin,native collagens and/or denatured collagen (gelatin). Preferably, theantimicrobial protein is selected from peroxidases (lacto, myelo andsalivary forms) and lysozyme.

In some embodiments, the substrate includes triglycerides.

In preferred embodiments, the liquid medium comprises water.

Embodiments of the invention are directed to compositions that includecomplexed angiogenin and native angiogenin. Preferably, the compositionincludes complexed angiogenin and native angiogenin at a ratio of 1:1 to1:10. More preferably, the composition also includes a buffer system, aphysiological acceptable base and a salt. Preferably, the buffer systemis oxalic acid, ethylenediamine tetraacetic acid or citric acid.Preferably, the physiological acceptable base is sodium bicarbonate.Preferably, the salt is sodium chloride, potassium chloride or calciumchloride.

Further aspects, features and advantages of this invention will becomeapparent from the detailed description of the preferred embodimentswhich follow.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other feature of this invention will now be described withreference to the drawings of preferred embodiments which are intended toillustrate and not to limit the invention.

FIG. 1: Isolation and Purification of ANG

Isolation of ANG using cation-exchange chromatography (UNO S6 column)with NaCl gradient (20% to 100% B) in 20 min. The time of injection ofANG sample (1 mL of 0.2 mg/mL in 20 mM sodium phosphate buffer pH 7.2)is indicated by the arrow. ANG eluted as two separate peaks at 39 min.(38% Buffer B) and 44.5 min. (60% Buffer B) respectively, from the timeof injection. ANG was detected by monitoring the absorbance at 214 nmusing a UV-Vis detector.

FIG. 2: Biotinylated ANG-LF Interactions

Interaction of biotinylated LF and biotinylated ANG with immobilized ANG() and immobilized LF (▴) was measured by kinetic ELISA. ANGimmobilized on a microtiter plate was incubated with biotinylated LF,vice versa; LF immobilized on a microtiter plate was incubated withbiotinylated ANG, respectively. Unbound proteins were removed bythorough washing with PBS-Tween buffer and the bound proteins weresubjected to an enzymatic reaction with avidin-alkaline phosphatase. Thedose dependence of rate of turnover of p-nitrophenyl phosphate substrateby avidin-alkaline phosphatase on the concentration of biotinylatedprotein indicated the interaction between LF and ANG.

FIG. 3. ANGex Detection by Turbidometry

Turbidity titration for the detection of ANGex in solution involvedmeasurement of A600 for a series of LF solutions to which increasingamounts of ANG was added. The relative light scattering as measured bythe absorbance at 600 nm, plotted against the molar concentration ratioof ANG to LF indicated non-linear increase in macromolecular size due tocomplex formation. Inset shows linear increase of A280 for the sameseries of solutions.

FIG. 4. Detection of ANGex Formation

Cation-exchange chromatogram is shown for the presence of ANGex, elutedusing 1 M NaCl gradient of (60-100% B in 80 min) and monitored by theabsorbance at 214 nm. (fdn)-LF (1 mg/mL) elutes as a single peak atretention time of 84.6 min (Curve 1). The retention time of this peakincreases upon ANGex formation with 1 mg/mL (Curve 2) and 4 mg/mL (Curve3) of ANG.

FIGS. 5A and B. Antioxidant Activity of ANG, LF and ANGex

Antioxidant activity was determined by FRAP assay with Vitamin C, TROLOXand FeSO₄ as standards, by following the increase in absorbance at 593nm with time. FIG. 5A shows the FRAP reaction kinetics data for LF, ANGand ANGex. The concentrations of the three standards, LF, ANG and ANGexwere 10 mg/ml (125 μM for LF, 694 μM for ANG). FIG. 5B compares theantioxidant efficiency (r) as measured by the change in absorbance withthe concentration of ANGex. Lines represent ANGex formed at varyingconcentrations of ANG with 0 (∘), 2 (), 6 (▾) and 10 mg/mL (▪) of LF.ANGex formed with 2 mg/mL of LF exhibits the highest antioxidantefficiency with r=0.046.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

While the described embodiment represents the preferred embodiment ofthe present invention, it is to be understood that modifications willoccur to those skilled in the art without departing from the spirit ofthe invention. The scope of the invention is therefore to be determinedsolely by the appended claims.

Suitable ANG can be isolated from dairy sources including colostrum,milk, whey and milk serum from humans, cows, buffalos, horses, sheep,pigs or camels. Additionally, ANG also can be purified from otherbiological fluids from animals (eg. blood), recombinant sources andgenetically-modified organisms (GMOs). Recombinant ANG may becloned-expressed in either prokaryotic or eukaryotic cellular systems.The ANG is isolated by any conventional method, such as by filtrationmethods, chromatography techniques using ion-exchanger, molecular-sieveor affinity columns.

Dietary supplementation with ANG may be helpful to individuals sufferingfrom vascular disorders such as congestive heart failure, myocardialinfarction, stroke, stable and unstable angina, poor circulation, forsupportive supplementation of long-term medications in the management ofhypertension, hyperlipidemia, diabetes, and chronic fatigue syndrome;mitochondrial diseases including mitochondrial encephalomyopathy, lacticacidosis, and stroke-like symptoms, Keams-Sayre syndrome and Alpers'disease.

ANG supplementation supports vascular health and promotes repair ofdamaged or clogged vascular tissue. However, in order to functionproperly, the structure of ANG must be stabilized.

The activity of ANG, like the activity of most proteins, is highlydependent on the three-dimensional or tertiary structure of the protein.If the protein does not have the proper conformation its activity isdiminished or lost. ANG's instability limits it usefulness. Milieuconditions such as metals (copper in particular), carbonic ions, salts,pH and conductivity affect the angiogenic properties of ANG. Inaddition, protein isolation procedures, storage, freezing-thawing, canadversely affect the biofunctionality of ANG. Consequently, before ANGcan be used for commercial application, it would be expected to becomedenatured or inactivated. (Soncin F, et al. Biochem Biophys Res Comm236:604-610, 1997)

Embodiments of the invention are directed to providing a stabilized formof ANG. ANG is immobilized on naturally occurring substrates. Suitablesubstrates include polysaccharides, proteins, lipids, nucleic acids,nucleotides, and vitamins. Preferred substrates include heparan sulfate,carrageenan, mucin, collagen, gelatin, fibronectin, transferrin,conalbumin, lysozyme, peroxidase, triglycerides, and vitamins.Galactose-rich polysaccharide (GRP), vitamin-E, ceruloplasmin,metallo-thionein and lactoferrin being the most preferred.

The attachment of ANG to the substrate may be non-covalent or covalentbinding. The interaction may be at the N-terminus, the C-terminus or anymolecular region or site of the ANG protein. In some embodiments, ANG isattached covalently to a polysaccharide, preferably a galactose-richpolysaccharide by covalent attachment to the N-terminus of ANG. In otherembodiments, the complex is formed by a non-covalent association betweenANG and a protein, lipid, nucleic acid, vitamin or carbohydratemolecule. The basis of the association may be electrostatic or byhydrophobic interaction or using bifunctional reagents.

ANG is immobilized on a naturally occurring substrate. Such substratesinclude organic compounds, which attach to the N-terminus domain of theANG protein. Most preferably, the substrate is a galactose-richpolysaccharide. Suitable galactose-rich polysaccharides includegalactose derivatives comprising galactose, anhydrogalactose,2-O-methyl-galactose, and 4-O-methyl-galactose, among others. The GRPsubstrates can be purchased or extracted from commercial agars by knownmethods. Other suitable biologically active substrates include proteins,such as collagen, denatured collagen (gelatin), fibronectin, and casein;polysaccharides, such as mucin, heparan sulfates, carrageenan, andcellulose; nucleic acids and their nucleotides, such as deoxyribonucleicacid and adenosine triphosphate; and lipids such as triglycerides.

ANG is immobilized on the substrate using any suitable technique. Forexample, ANG can be immobilized simply by mixing the ANG with thebiologically active substrate in a suitable medium, such as deionizedwater. The immobilization process is dependent on the quality of thesubstrate as well as the quality of the ANG. Consequently, the amount ofsubstrate and the amount of ANG to be used in the immobilizationreaction will depend, inter alia, on the choice of starting materials.The immobilization technique and the amounts of substrate and ANG willbe readily determinable by a skilled artisan without undueexperimentation.

ANG complexes could be formed with a second protein based on functionalassociation or a synergy that may enhance ANG function. The ANG proteincomplexes could be formed by physical, charge and/or chemicalinteractions. ANG and a protein substrate may be complexed togetherdirectly or they may be complexed by means of an appropriatebifunctional reagent. A non-covalent complex may be formed by means ofelectrostatic interactions which may be enhanced by inclusion ofappropriate buffers and/or salts.

Embodiments of the invention include methods such as biotin-avidinbinding and disulfide bonding. The ANG polypeptide chain consists of 6cysteine residues that form 3 disulfide bonds at 26-81, 39-92, and57-107 residue positions. ANG may be labeled with biotin and mixed witha substrate protein associated with avidin to form a complex byassociation of the biotin with the avidin. Alternatively, the ANG may beattached to avidin and the biotin complexed onto the substrate protein.

The formation of a complex may be confirmed by usingco-immunoprecipitation techniques. In preferred embodiments, the proteinsubstrate is from the group including, but not limited to transportproteins, subepithelial matrix proteins and antimicrobial proteins.Transport proteins include but are not limited to lactoferrin,transferrin, ovo-transferrin (conalbumin), ceruloplasmin,metallo-thionein and transfer factors. Subepithelial matrix proteinsinclude but are not limited to fibronectin, fibrinogen, laminin,vitronectin, osteopontin, native collagens and denatured collagen(gelatin). Antimicrobial proteins include but are not limited toperoxidases (lacto, myelo and salivary forms) and lysozyme.

Embodiments of the invention are directed to ANG complexed with lipids.ANG may be formed into a complex with CoQ-10 by physical and/or chemicalinteractions. ANG and CoQ-10 may be complexed together directly or theymay be complexed by means of an appropriate bifunctional reagent. Anon-covalent complex may be formed by means of electrostaticinteractions which may be enhanced by inclusion of appropriate buffersand/or salts.

In some embodiments, ANG is incorporated into anionic lipid films byelectrostatic interactions. ANG may be complexed into micro-emulsions.Lipids used to form such films and micro-emulsions includetriglycerides, phospholipids including commercially availablepreparations such as Phospholipid Lipoid S 100 (Lipoid KG, Germany),lipophilic vitamins such as Coenzyme-Q10, Vitamin A, Vitamin E andUbiquinone. Other Vitamins which complex with ANG include nicotinamideadenine dinucleotide (NADH) and Ascorbic acid. More preferably, thephospholipid is one or more selected from Docosahexaenoic acid (DHA),phosphatidyl glycerol, phosphatidyl inositol, phosphatidyl serine,phosphatidyl choline, phosphatidyl ethanolamine, phosphatidic acids,ceramides, cerebrosides, sphingomyelins and cardiolipins.

Nucleic acid/nucleotide-based ANG complexes may be formed based uponelectrostatic interations between the positively charged ANG and thenegatively charged DNA or RNA. The formation of such complexes isconfirmed by Gel Mobility Shift Assay (Bading H. Nucleic Acid Research16: 5241-5248, 1988).

In an embodiment of the present invention, ANG may be combined withmetal ions such as copper and zinc, preferably copper.

In some embodiments, ANG is used as an aqueous solution containing amixture of the ANGex and native ANG, where the concentration of themixture in the solution is from about 0.001 to about 2.5% wt/vol and theratio of ANGex to native ANG in the mixture is from about 1:1 to about1:10, preferably about 1:1 to 1:5, and most preferably about 1:1. And insome embodiments, the mixture contains about 1% wt/vol ANGex and about1% wt/vol native ANG.

In some embodiments, the aqueous solution further includes a buffersystem that contains a physiologically acceptable acid, such as oxalicacid, ethylenediamine tetraacetic acid, and citric acid, preferablycitric acid, a physiologically acceptable base, preferably sodiumbicarbonate, and a physiologically acceptable salt, such as calciumchloride, potassium chloride and sodium chloride, preferably sodiumchloride. The molar ranges of acid:base:salt is generally about 0.1 to0.0001M (acid):1 to 0.001M (base):10 to 0.01M (salt); with 0.01-0.001M(acid):0.1 to 0.01M (base):1 to 0.01M(salt), preferred; and 0.001M(acid):0.01M (base):0.1M(salt), most preferred.

ANG useful in accordance with the present invention includes ANGisolated from mammalian sources (humans, cows, sows, mares, transgenicanimals and the like), biological secretions such as serum, colostrum,transitional milk, matured milk, milk in later lactation, and the like,or processed products thereof such as skim milk and whey. Also useful isrecombinant ANG cloned-expressed in either prokaryotic and eukaryoticcells. ANG is isolated by any conventional method, such as bychromatography, ion-exchanger, molecular-sieve or affinity column. In apreferred embodiment, ANG is isolated from plasma, serum, milk or a milkproduct. Despite their low abundance, ANG is easily isolated from milkor serum because it is more basic than other RNases. In a particularlypreferred embodiment, ANG is co-isolated with lactoferrin from milk ormilk product (Bond, et al., Biochemistry 27:6282-6287, 1988). TheANG/lactoferrin isolate is then stabilized as described herein.

Without intending to be limited by a theory of operation, it is believedthat immobilization gives structural stability and bio-functionalspecificity to ANG. It is expected that ANGex molecules will demonstratea molecular orientation similar to its orientation when adhered toendothelial and fibroblast cells when forming the extra-cellular matrixwhich is part of the angiogenic process. This property facilitates theretention and carry-through of ANG.

In preferred embodiments, the ANGex is combined with native ANG. Themolecular ratio of ANGex versus native ANG is important in thespecificity, broad-spectrum activity, and molecular stability of boththe immobilized and the native ANG. Mixtures of ANGex and native ANG ina ratio of from about 0.25:1 to about 1:10, preferably from about 1:1 toabout 1:2 of native ANG to ANGex, most preferably 1:1 ratio arepreferred.

Mixtures of ANGex and native ANG are formed by adding excess ANG to thesubstrate. In a representative embodiment, from about 0.001% wt/vol toabout 2.5% wt/vol, preferably from about 0.5% wt/vol to about 2.0%wt/vol, most preferably about 1% wt/vol of ANG is added to a solutioncontaining 0.01% wt/vol galactose-rich polysaccharide.

In a preferred embodiment, the aqueous solution is buffered with acombination of a physiologically acceptable acid, such as oxalic acid,ethylenediamine tetraacetic acid, or citric acid, preferably citricacid, a physiologically acceptable base, preferably sodium bicarbonate,and a physiologically acceptable salt such as calcium chloride,potassium chloride or sodium chloride, preferably sodium chloride. Thecitrate and bicarbonate ratio in the buffer is significant forco-coordinated metal binding properties of ANG. The molar ranges ofacid:base:salt is typically about 0.1 to 0.0001M (acid):1 to 0.001M(base):10 to 0.01M (salt); with 0.01-0.01M (acid):0.1 to 0.01M (base):1to 0.1M(salt), preferred; and 0.001M (acid):0.01M (base):0.1M(salt),most preferred.

ANGex may be substituted for ANG for any treatment for which ANG isuseful. ANGex brings the added benefits of increased stability for ANGas an active agent. Because of the increased stability, ANGex hasincreased residence time compared to ANG so that both dosage andfrequency of administration is less than with ANG.

ANGex is useful to improve cardiovascular function due to its well knownability to promote formation of new blood vessels. In addition, ANGexhas antimicrobial and antioxidant properties as shown herein.

ANGex may be useful in treatment of cardiovascular disease,gastrointestinal disorders, maintenance of bone and joint health,maintenance of cognitive health, oropharyngeal health. ANGex may havecosmetic uses such as hair growth and regeneration, anti-aging,reduction of wrinkles and age spots, and skin rejuvenation. ANGex may beuseful in the treatment of acne and other skin disorders includingfolliculitis, furunculosis, and TED. ANGex may be useful inpost-operative recovery including surgical wound repair/healing, tissueresurfacing, and plastic surgery, both cosmetic and reconstructive.ANGex may be useful as a coating on biomaterials including sutures,implants, indwelling catheters and dental floss. ANGex may also beuseful in veterinary practices and for increasing weight gain in meatanimals.

EXAMPLES Example-1 Angiogenin (ANG) Preparations

Bovine milk (6% fat, 3% protein) was concentrated 2-3 fold, byultra-filtration through a polysulfonic membrane (pore size 30 kDa). ANGwas isolated from the ultra-filtrate by precipitation of proteins withammonium sulfate, followed by cation-exchange chromatography aspreviously described (Fedorova T V, et al., Appl. Biochem. Microbiol.38:193-196, 2002). Briefly, the protein precipitated with 60% ammoniumsulfate was dissolved in 0.01 M potassium phosphate buffer (pH 6.7) anddialyzed against the same buffer at 4° C. for 36 h. The dialysate wasloaded on to a CM-cellulose 52 column (Serva, Germany) and 4-mLfractions were collected. Fractions with A280 ≧0.1 were pooled anddialyzed against 10 mM Tris-HCl buffer (pH 8.3). The dialyzate was thenloaded on a CM-Toyopearl 650 S column (Tosoh, Japan) equilibrated with0.05 M Tris-HCl buffer (pH 8.3). Column-bound proteins were eluted witha linear gradient of KCl concentration in the same buffer at a flow rateof 0.5 mL/min. ANG-containing fractions were further pooled, dialyzedagainst a 0.01 M potassium phosphate buffer (pH 7.0), and freeze-dried.

Furthermore, a second preparation of bovine milk ANG (mixture of RNAsetype-4 and -5 proteins) obtained from Tatua Nutritionals (Morrinsville,New Zealand) was also used in the immobilization experiments. Thissecond preparation of ANG (initially enriched from skim milk along withall other positively charged milk proteins) was isolated bycation-exchange chromatography. The column bound proteins were elutedstep-wise by using various concentrations of salt (NaCl). The ANG-richfraction was de-salted and concentrated by ultra-filtration. ThisANG-rich preparation demonstrated >40% RNAse activity. For experimentalpurposes, the ANG-rich fraction was further purified to a higher levelof purity, as follows: the ANG-rich fraction was run on a second type ofcation-exchange column and protein was eluted with increasingconcentrations of NaCl and by increasing the pH. This process separatedANG into (RNAse 4)-rich and (RNAse 5)-rich fractions, which wereultra-filtered and freeze-dried into powder form.

The purity and homogeneity of both lab-scale and pilot-scale ANGpreparations was assessed by cation-exchange chromatography. The samplewas loaded on to a UNO S6 column (Bio-Rad) equilibrated with 20 mMsodium phosphate buffer at pH 7.2. After extensive washing with the samebuffer, ANG was eluted from the column by a gradient flow of 20 mMsodium phosphate buffer containing 1 M sodium chloride (20 to 100% in 20min). Molecular weight of ANG (17 kDa) was confirmed by SDS-PAGE.

FIG. 1 depicts the isolation and purification of ANG using UNO S6cation-exchange column. The time of injection of ANG sample (1 mL of 0.2mg/mL in 20 mM sodium phosphate buffer pH 7.2) is indicated by thearrow. ANG eluted as two separate peaks at 39 min. (38% Buffer B) and44.5 min. (60% Buffer B) respectively, from the time of injection. ANGwas detected by monitoring the absorbance at 214 nm using a UV-Visdetector.

Reconstitution of ANG Protein in Buffered Solutions

For immobilization on different substrates, ANG was reconstituted intosolution at a protein concentration of 1 mg/mL. The following differentbuffer solutions compatible with the chemical nature of theimmobilization/conjugation substrates were used: i) carbohydrate-basedsupport systems, 0.1 M sodium bicarbonate buffer [with 0.5 M NaCl (pH8.3)]; ii) protein-based support systems, 20 mM sodium phosphate buffer(pH 7.2) or citrate-bicarbonate buffered saline [(CBS), pH 8.0;consisting of 1 mM citric acid, 10 mM NaHCO₃, and 100 mM NaCl]; and iii)lipid-based support systems, 50 mM sodium phosphate buffer (pH 7.0).

Preparation of Biotinylated ANG Reagents

ANG was biotinylated using biotin disulfide N-hydroxysuccinimide ester(Sigma), which contains an active ester to react with the primary amineof ANG. This reagent was dissolved in N,N-dimethylformamide at aconcentration of 25 mg/mL. ANG (20 mg) was dissolved in 2 mL of 0.1 Msodium phosphate buffer, pH 7.6 to give a final concentration of 10mg/mL. Reagent solution (volume: 0.4-mL volume) was slowly stir-mixedwith the ANG solution, such that a 15 molar excess of reagent is presentin the mixture. The mixture was gently agitated on a rocker for an hourat room temperature. The biotinylated protein was separated from excessreagent by gel filtration on a Sephadex G-25 column. The column wasequilibrated with 10 mM PBS containing 138 mM NaCl and 2.7 mM KCl at pH7.4. The mixture was loaded on to the column and washed with few dropsof PBS. Biotinylated ANG (b-ANG) was eluted with PBS in 0.5 mL fractionsmonitoring the A280 of the eluent to confirm the presence of b-ANG. Allthe b-ANG fractions were pooled, its concentration was determined byBradford assay and the degree of biotinylation was estimated byHABA/Avidin Assay.

Example-2 Preparation of ANGex Using Carbohydrate-Based Substrates

ANG is immobilized on carbohydrate-based supports using reductiveamination process that involves reactive aldehyde groups generated bypartial hydrolysis of agarose (or agar). Acid hydrolysis of agar undermild conditions leads to cleavage of glycosidic bonds between C-1 of3,6-anhydro-L-galactose and O-3 of D-galactose residues that exposesD-galactosyl residues and leads to the formation of galactose-richpolysaccharides (GRP) (Gray G. Arch. Biochem. Biophys. 163:426-428,1974; Lee R T, et al., Biochemistry 19:156-163, 1980).

Galactose-rich polysaccharide (GRP), extracted as a water-solublefraction from agar was used as the substrate for ANG immobilization. GRPwas prepared by adding 1-g of bacteriological agar (Difco) to 10-mL ofcitrate-bicarbonate buffered saline [(CBS), pH 8.0; consisting of 1 mMcitric acid, 10 mM NaHCO₃, and 100 mM NaCl]. After thorough mixing on avortex, the mixture was centrifuged for 2 min at 2500 rpm to obtain theGRP supernatant. The clear GRP solution was carefully aspirated and usedas the substrate for ANG immobilization.

Other suitable polysaccharide substrates for ANG immobilization includesulfated glycosaminoglycans (GAG), i.e. heparin and heparan sulfate;various types of carrageenan substrates, i.e., Satiagel® brands(Degussa) ADG-14 (kappa/iota-type), ADF-23 (kappa/iota-type), DF-52(iota-type); Genulacta® brand (Hercules) 1M K-100 (kappa-type); andCarravisco® brand DFL-1 (lambda-type). The binding mechanism is a simpleelectrostatic interaction between the positive amino groups on the ANGprotein and the negative sulfate groups of the GAG and carrageenan.

Accordingly, a carbohydrate substrate (i.e. GRP, carrageenan, GAG oragarose) is thoroughly washed in distilled water, suspended in 0.2 M HCl(2-3 mL/g moist gel) with gentle agitation. After incubation at 55° C.for 3 h, the gel suspension is neutralized with dibasic sodium phosphate(0.2 M) solution, and washed with sodium phosphate (0.1 M) buffer at pH7.2. The moist gel is filtered by gentle suction, weighed andreconstituted to a known concentration in buffer (ca. 75 mg/mL). Thepartially hydrolyzed gel is stored on ice and used within 3 hours ofpreparation. ANG (protein concentration: 1 mg/mL) was dissolved in 0.1 Msodium bicarbonate buffer with 0.5 M NaCl, pH 8.3. For immobilization, a4 mL suspension containing 0.3 g moist gel in buffer solution was mixedwith ANG solution and 6 mL of 100 mg/mL cyanogen bromide solution inDioxan (150 mg of CNBr/mL of Sepharose®). The reaction mixture wasgently agitated at 25° C. for 1 h, loaded on to a column and the gel (orbeadlets) was extensively washed with coupling buffer (at least 5-6column volumes) until no more amino groups (or excess protein) wasdetected in the washings.

In another method, 0.3 g of moist gel was suspended in 4 mL of bufferand was mixed with ANG solution and freshly prepared sodiumcyanoborohydride solution (80 mM). The mixture was agitated at roomtemperature for 2 h and loaded on to a column and the gel (or beadlets)was extensively washed with coupling buffer (at least 5-6 columnvolumes) until no more amino groups (or excess protein) was detected inthe washings.

In order to block any remaining active groups, the gel is transferred to10 mL of 0.1 M Tris-HCl buffer, pH 8.0 and allowed to stand for 2 h. Thegel (or beadlets) was washed again with 3 alternative cycles of high andlow pH buffers. Each wash cycle consisted of at least 5 column volumesof 0.1 M acetic acid/sodium acetate, pH 4.0 containing 0.5 M NaCl;followed by a wash with 5 column volumes of 0.1 M Tris-HCl, pH 8.0containing 0.5 M NaCl. This procedure ensured that no free ligand (ANG)bound non-specifically to the gel.

Example-3 Preparation of ANGex Using Protein-Based Substrates

ANG was immobilized on protein substrates via non-covalent interactionsand conjugation methods including biotin-avidin affinity binding anddisulfide bonding techniques. Suitable functional protein substrates: i)physiological transport-proteins including but not limited tolactoferrin, transferrin, ovo-transferrin (conalbumin), ceruloplasmin,metallo-thionein and transfer factors; ii) sub-epithelial matrixproteins including but not limited to fibronectin, fibrinogen, laminin,vitronectin, osteopontin, native collagens and denatured collagen(gelatin); iii) antimicrobial proteins including but not limited toperoxidases (lactoperoxidase, LPO; myeloperoxidase, MPO; and salivaryperoxidase, SPO) and lysozyme.

Measurement of Biotinylated-ANG and LF Interactions by ELISA.

For binding studies, a 1% LF substrate (cow milk protein isolate) wasprepared in 20 mM sodium acetate buffer (pH 4.0). A 1% LF solution(volume: 0.1-mL) was added to each well of a microtiter plate (CorningCostar® 3690) and allowed for an overnight surface adsorption at 4° C.The microplate was washed extensively with acetate buffer to remove anyunbound LF. A 0.5% Tween (in PBS) was added to each well to block anyunbound surface. Various concentrations (ranging 0 to 1 mg/mL) ofbiotinylated ANG solution (in 20 mM PBS, pH 7.2) was added to individualwells and further incubated at 4° C. overnight, to achieve equilibrium.Any unbound ANG was removed by repeated washing with PBS-Tween.Interactions between ANG and LF were measured by using avidin-conjugatedalkaline phosphatase reagent, followed by a color reaction with theaddition of p-nitrophenyl phosphate (PNP), a chromatophore substrate.The rate of color development was measured at 405 nm, in a kineticmanner for 30 min. The rate of enzymatic hydrolysis of PNP chromatophoreas function of concentration of biotin-ANG was plotted to measure thebinding interactions.

FIG. 2 depicts the interactions of biotinylated LF with (fdn)-ANG(subset-A), and biotinylated ANG with (fdn)-LF (subset-B), respectively,as measured by kinetic ELISA. The rate of PNP substrate hydrolysis byavidin-alkaline phosphatase, as measured by kinetic ELISA, indicated theinteraction of biotinylated protein(s) with (fdn)-ANG or -LF. Data showsthe formation of ANG-LF complexes in a dose-dependant manner.

Example-4 Preparation of ANGex Using Lipid-Based Substrates

Based on its cationic properties, ANG is incorporated into the anioniclipid films and matrices by electrostatic interactions. A fattyacid-like stearic acid (Octadecanoic acid C18H36O2) is thermally coatedunder vacuum on a quartz cover slip or quartz crystal resonator of thetype used in Quartz Crystal Microbalance (QCM) technique. This processis known to produce a film of few hundred angstrom thickness. Thestearate-coated quartz cover slip is immersed in 0.1% ANG solution (in50 mM sodium phosphate buffer) at 4° C. for 30 min. The quartz coverslip is removed from the ANG solution, washed thoroughly in deionizedwater and dried under gentle flow of dry nitrogen gas.

Measurement of ANG Interaction with Lipid Film by QCM

Immobilization of ANG on a thermal evaporated fatty acid film wasmonitored by measuring changes in the frequency of quartz crystals usingQCM instrument (Edwards FTM5 microbalance) at 1 Hz resolution. Thefrequency changes are converted to mass loading using the Sauerbreyequation. The quartz cover slip is also used for recording the UVabsorption spectrum of the film before and after the lipidimmobilization of ANG. The difference in the UV spectrum indicated theimmobilization of ANG on the lipid film (Sauerbrey G., Z. Phys. 155:206,1959; Buttry D A, et al., Chem. Rev. 92:1356-1379, 1992; Sastry M.,Trends in Biotechnology 20:185-188, 2002).

ANG was immobilized and entrapped in the lipid matrix primarily byelectrostatic interactions. The use of charge interactions also enabled“leaching-out” of the entrapped ANG when immersed in solution at pH 2.0.Soft lipid matrix enabled the entrapment without significant distortionof the tertiary structure of the ANG, therefore, the bio-functionalproperties of the protein molecule is well preserved.

Example-5 Preparation of ANGex Using Coenzyme/Vitamin-Based Substrates

Coenzyme-Q10 (CoQ-10), due to its quinone structure, is extremelylipophilic, soluble in ethanol and chloroform, but practically insolublein water. A detergent like sodium glycocholate along with sonicationgenerates a submicron-sized dispersion of CoQ-10 in phospholipid (fromsoy bean). The detergent also inhibits re-crystallization of CoQ-10.This dispersion has been used in the preparation of micro-emulsions toincorporate ANG (Stojkovic M, et al., Biol. Reprod. 61:541-547, 1999).

Phospholipid Lipoid S 100 (Lipoid KG, Germany) is a mixture ofphosphatidylcholine from fat-free soybean lecithin that consists mainlyof linoleic phosphatidylcholine. S 100 was melted together with CoQ-10(Alchem, India) at 65° C. Sodium glycocholate (NaGC) was dissolved indistilled water containing 2.25% (w/w) glycerol. This mixture was heatedto 65° C. and mixed with the molten lipid phase. The hot mixture wassonicated for 30 min. while maintaining a constant temperature. This hotemulsion was sterile-filtered (0.22 μm) into vials and allowed to coolat room temperature. Stabilizer dispersion without CoQ-10 was alsoprepared in a similar manner. Calculated amounts of the two dispersionswere mixed to prepare dispersions containing various concentrations ofCoQ-10.

ANG stock solution was freshly prepared using 50 mM phosphate buffer, pH8.0, containing 0.2% sodium cholate. This solution was diluted with 50mM phosphate buffer, pH 7.4, containing 0.2% sodium cholate to a proteinconcentration of 1 mg/mL. The ANG-CoQ-10 complexes were prepared bymixing 500 μL aliquots of diluted ANG solution with 500 μL of ubiquinonedispersions at various concentrations up to 100 μM. The mixtures werekept in an incubator-shaker at 25 C for 1 h, before characterization byantioxidant assay.

ANG-NADH complex was prepared by mixing 500 μL of ANG (1 mg/mL) solutionwith 500 μL of β-NADH-dipotassium salt (0.1 mg/mL) solution in 50 mMphosphate buffer, pH 7.4.

Ascorbic acid has pKa value of 4.2 and would complex with ANG. Thiscomplex was prepared by mixing 500 μL of ANG (1 mg/mL) solution with 500μL of ascorbate solutions of different concentrations in 50 mM phosphatebuffer, pH 7.4.

Vitamins A and E are also fat-soluble and their complex with ANG wasalso prepared by dispersion in soy phospholipids, as described withCoQ-10 complexes

Example-6 Preparation of ANGex Using Nucleic Acid/Nucleotide-BasedSubstrates

DNA, RNA and nucleotides are negatively charged due to the presence ofphosphate groups. These molecules form complexes via the positivelycharged residues on basic proteins such as ANG. The characterization ofthe affinity and shape of the ANG-nucleotide complex is performed usingGel Mobility Shift assay (Bading H. Nucleic Acids Research 16:5241-5248,1988). The DNA-binding assay using mobility-shift polyacrylamide gelelectrophoresis (PAGE) is based on the observation that protein/DNAcomplexes migrate through polyacrylamide gels more slowly than unboundDNA fragments.

ATP-protein complex was formed by the treatment of the ANG with ATP(Sigma). ATP (10 μmoles) was incubated with ANG (5 mg) and 40 μMTris-acetate buffer, pH 7.0, in a total volume of 1 mL for 10 min atroom temperature. The complex was centrifuged, washed, and theATP:Protein ratio determined (Richardson S H, et al., Proc. Natl. Acad.Sci. USA 50:821-827, 1963).

Example-7 Preparation of [ANGex]+[(Fdn)-ANG] Mixtures

The molecular ratio of ANGex versus (fdn)-ANG is important for thespecificity, multi-functionality and molecular stability of both theimmobilized and the native ANG proteins. Mixtures of ANGex and (fdn)-ANGare formed by adding excess (fdn)-ANG during the immobilization,conjugation or complexation process. Mixtures of ANGex and (fdn)-ANG ina ratio of from about 100:1 to about 1:100, preferably from about 10:1to about 1:10, most preferably 1:1 ratio have been found to provide theoptimum stability and functionality.

Example-8 Detection of ANGex

The formation of ANGex was confirmed by a turbidity titration method,commonly used to study formation of several macromolecular complexessuch as protein-DNA and protein-polyelectrolyte complexes. Duringformation of ANGex (i.e. ANG+LF complex), the interaction is accompaniedby an increase in the macromolecular size that results in the increaseof incident light scattering. The relative light scattering isquantified by spectral analysis as apparent absorbance at 600 nm, whilesubtracting any interfering absorbance from the sample or buffer (ZhouY, et al., Biophys Chem 107:273-281, 2004; Xia J, et al., Langmuir9:2015-2019, 1993).

FIG. 3 shows the detection and measurement of ANGex by turbiditytitration method. The assay is based on measurement of A₆₀₀ for a seriesof LF solutions to which increasing amounts of ANG was added. ANGex(ANG+LF complex) was prepared in a series of samples with varyingamounts of ANG (0 to 25 μM) with LF (5 μM) solution in 20 mM phosphatebuffer. The protein absorbance at 280 nm showed a linear increase withthe molar ratio [ANG]/[LF] of the complex forming proteins. The relativelight scattering as measured by the absorbance at 600 nm, plottedagainst the molar concentration ratio of ANG to LF indicated non-linearincrease in macromolecular size due to ANGex complex formation. (Inset)shows linear increase in A₂₈₀ absorbance for the above series ofsolutions.

Isolation of ANGex

ANGex with carbohydrate or protein substrates are isolated bysize-exclusion and/or ion-exchange chromatography. Due to larger size,ANGex is eluted earlier than the free proteins or free carbohydrates onthe size-exclusion column. A size-exclusion column (TSKgel G4000PW,Tosoh Biosep, Japan) was equilibrated with 20 mM sodium phosphatebuffer, pH 7.2. The ANGex solution was injected into to the column andsubsequently eluted with sodium phosphate buffer at a flow rate of 0.5mL/min.

During isolation with cation exchange column (UNO-S; Bio-Rad), asolution containing ANGex was applied to a cation exchange column whichwas equilibrated with 20 mM sodium phosphate buffer (pH 7.2). A flowrate for the buffer was continued at 1 mL/min for 30 min to ensureremoval of unbound chemical residues from the ANGex solution. The boundANGex was eluted by flow of salt gradient (1 M NaCl in phosphatebuffer). In the case of ANG-LF complex, free proteins and the complexwere eluted as separate peaks at distinct salt concentrations. However,for ANG-GAG complex, a combination of a cation-exchange and an anionexchange columns were used in series, since such complex shows residualnegative charge from the GAG component and binds to anion-exchangecolumn along with free GAG. On the other hand, the free protein due toits positive charge binds to the cation-exchange column. The boundcomplexes are eluted using a salt gradient flow separately fromrespective columns.

FIG. 4 shows the separation of ANGex from free LF, on UNO-Scation-exchange chromatogram using NaCl gradient (60-80% B in 80 min).Unbound (fdn)-LF (50 μM) eluted as a single peak at retention time of123.3 min (Curve 1). The retention time of (fdn)-LF peak decreased to121.6 min for ANGex with ANG/LF molar ratio 1.0 (Curve 2). Elution ofANGex was monitored by measuring absorbance at 214 nm.

Example-9 Molecular Stability of ANGex

The three-dimensional structure of ANG at the physiological pH andtemperature conditions is responsible for it multi-functional activity.Changes in milieu conditions such as the pH, ionic strength, as well asinteraction of ANG with other proteins, ligands and denaturantsinfluence the structure-functional properties of the native state.Higher temperatures also lead to unfolding of the native structureleading to diminished or total loss of ANG activity. Data on changes inthe secondary structure of ANG were obtained using Circular Dichroism(CD) spectrophotometric assay. ANG polypeptide backbone is opticallyactive in the far ultraviolet region (170-250 nm) and differentsecondary structures exhibit characteristic CD spectra.

In an experiment to study the acid-tolerance/resistance of ANGex, aseries of samples were prepared with 10 μM of ANGex, dissolved in 1-mLof buffers with varying pH ranging from 2.0 to 8.0. After incubation at37° C. for 30 min with gentle shaking, the CD spectrum of each of thesample was recorded using a CD spectrophotometer (Jasco, Japan).Comparison of the CD spectra of ANGex samples in the range 190-240 nm,with that for ANGex at pH 7.2 revealed only minor changes in thesecondary structure of the complex. This observation confirmed thatANGex is stable to changes in pH of the solution.

A similar experiment was carried out in order to assess thethermo-stability of ANGex. A series of ANGex samples (10 μM) in 50 mMacetate buffer, pH 5.5, were incubated at different temperatures (50, 60and 70° C.) for various time points (5, 10, 30, and 60 min). Afterincubation, the CD spectrum of each of the sample was recorded using aCD spectrophotometer equipped with a thermostatic cell holder. Therewere no significant changes in the CD profile of ANGex at highertemperatures when compared with the profile at room temperature,confirming that ANGex remained its native structure at elevatedtemperatures.

Measurement of ANG Activity by Placental RNase Inhibitor (PRI) Assay

For both acid and thermal stability testing protocols, the residualactivity of ANGex was monitored by an in vitro binding assay for ANGusing PRI (Bond M D., Anal. Biochem. 173:166-173, 1988). In brief, testsamples (volume range: 0 to 40 μL) were added to 40 μL 0.5 M Trisbuffer, pH 7.5 (with 5 mM EDTA, 10 mM DTT, 0.5 mg/mL human serumalbumin) containing of 0.53 pmol PRI (Sigma). The mixtures were made upto a final volume of 90 μL with deionized RNase-free water, incubatedfor 5 min for optimal interaction between ANG and PRI, followed by anaddition of RNase A (0.58 pmol of RNase A in 10 μL of 5 mM Tris, 0.1mg/mL lysozyme, pH 7.5). The reaction was initiated by the addition of0.1-mL of 1% yeast RNA (freshly dissolved in RNase-free water and passedthrough a sterile 0.45 μm filter). After incubation for 25 min at 25°C., the reactions were terminated with 0.2-mL ice-cold quenching reagent(1.16 N perchloric acid with 5.9 mM uranyl acetate) and the mixture washomogenized and placed on ice for 25 min. After centrifugation at 4° C.(15000×g for 5 min.), test samples were made from 0.2-mL aliquotsdiluted to 1-mL volume with 5 mM Tris, pH 7.5 containing 0.1 mg/mLlysozyme (in order to minimize adsorption to container surfaces). Theabsorbance at 260 nm was recorded for each sample and subtracted fromthat of a blank solution prepared in an identical manner except thatwater was added instead of RNase, to give ΔA₂₆₀.

A linear relationship between the RNase concentration and increasedabsorption at 260 nm due to the presence of the acid-soluble nucleotidesproduced from the hydrolysis of the yeast RNA, is observed. Two types ofstandards were used with each binding assay, i) containing 0.58 pmol(2.9 nM) RNase A in the absence of PRI or ANG (ΔAE); and ii) containingboth the RNase A and 0.53 pmol (2.6 nM) PRI (ΔAEI). These standardsrepresent the maximum and minimum obtainable ΔA₂₆₀ values, respectively,in the absence of ANG in the sample. Increases in ΔA₂₆₀ values above theminimum are proportional to the amount of ANG present, and theconcentration is calculated using the equation:[ANG]=[(ΔAsample−ΔAEI)/ΔAE]×2.9 nM. Wherein, ΔAsample, ΔAEI and ΔAE areabsorbance values of the sample, the RNase A plus PRI standard assay,and the RNase A standard, respectively. In particular cases, when impuresamples are assayed, concentrations are expressed in terms of RNase Aequivalents in μg/mL instead of nanomolar ANG concentration. Acomparative activity of ANG, before and after incubation at differenttemperatures as well as incubation in buffers at various pH, are used inthe estimation of acid-tolerance/resistance and thermo-stability ofANGex and ANG samples.

Example-10 Measurement of Functional Activities of ANGex RNase Activityof ANGex

RNase activity of ANG is far lower than that of RNase A (Ribonuclease A)and therefore a highly sensitive assay was used to measure thisenzymatic activity. This assay monitors the kinetics of cleavage offluorescent tagged oligonucleotides (substrates) by ANGex and measuresthe fluorescence emission following the cleavage (Park C et al.,Biochemistry 41:1343-1350, 2002).

The RNase substrates are short oligonucleotides of sequences (AUAA,AAUAAA, AAAUAAAA) each attached to a fluorophor, 6-carboxy fluorescein(6-FAM) at the 5′ end and to a quencher, 6-carboxytetramethyl-rhodamine(6-TAMRA) at the 3′ end. When the substrate is intact, 6-TAMRA quenchesthe fluorescence of 6-FAM. Ribonucleolytic cleavage of the substrateresults in the fluorescence emission by 6-FAM. RNase substrate (6 nM)solution was prepared in 1 mM Bis-Tris buffer, pH 6.0 containing 0.1MNaCl. A 2-mL substrate solution was mixed with 50 nM ANGex and tested at25° C. Fluorescence emission of the reaction mixture was measured at 515nm with excitation at 490 nm using a fluorescence spectrophotometer(QuantaMaster 1, Photon Technology International, NJ). Values of ΔF/Δtwere determined by a linear least squares regression analysis of theinitial fluorescence (F) with time (t). This rate was further used inthe following equation: kcat/KM={(ΔF/Δt)/(Fmax−F0)}·(1/[E]), where [E]is the concentration of ANGex. Fmax is obtained from a separateexperiment by adding 0.1 μM RNase A to the reaction mixture and F0 isthe initial fluorescence intensity without ANG.

A comparison of the kcat/KM for (fdn)-ANG with that of ANGex preparedfrom different substrates indicated the effect of immobilization.

Antimicrobial Activity of ANGex

Microbial metabolism cause electrical charge alterations in cultivationmedia due to breakdown of nutrients. A Bactometer® Microbial MonitoringSystem Model-128 (bioMerieux Vitek, Hazelwood, Mo.) was used to monitorbacterial growth by measuring impedance signals (a function of bothcapacitance and conductance) in the cultivation media. Growth ImpedanceDetection Assay (GIDA). Growth Impedance Detection Assay (GIDA) wasperformed in 16-well modules; briefly, a volume of 0.5-mLdouble-strength tryptic soy broth (2×TSB) was added to each well. Avolume of 0.25-mL of test sample (final concentration: 1 mg/mL) followedby 0.25-mL of bacterial suspension (104 cells/mL) prepared in 0.9%saline was added to the wells. Addition of 0.5-mL saline or bacterialsuspension to module wells with 0.5-mL TSB (2×) served as controls forsterility and growth, respectively. The inoculated modules (finalvolume: 1-mL) were incubated at 37° C., and impedance changes in themedia was continuously monitored by the Bactometer® at 6-min intervalsfor 48-h. Bacterial growth curves were graphically displayed as percentchanges of impedance signals versus incubation time. The amount of timerequired to cause a series of significant deviation from baselineimpedance value was defined as the ‘detection time’ (DT). Difference inDT values between growth control and test samples was considered as the‘stasis’ (growth-inhibition) time.

The antimicrobial activity of ANGex (immobilized on GRP) was comparedwith that of (fdn)-ANG and GRP (galactose-rich polysaccharide from agar)activities, tested at 1 mg/mL concentration. ANG demonstrated ability toinhibit the growth of various microbial pathogens. The immobilizationprocess of the free dispersed native ANG with GRP substrate resulted inthe generation of an ANGex complex with enhanced microbial growthinhibition activity as shown in Table-1.

TABLE 1 Antimicrobial activity of ANG and ANGex (immobilized on GRP)IMPEDENCE DETECTION TIME (Stasis) TEST STRAIN Control (h) ANG ANGexEscherichia coli ATCC433895 5.1 9.0 (3.9-h) 11.4 (6-3-h) Salmonellatyphimurium 6.2 8.6 (2.4-h) 10.5 (4.3-h) ATCC14028 Pseudomonasaeruginosa 12.5 28.7 (16.2-h)  36.0 (23.5-h) ATCC27853 Klebsiellapneumoniae 7.2 11.4 (4.2-h)  14.5 (7.3-h) ATCC13883 Staphylococcusaureus 11.8 16.6 (4.8-h)  21.4 (9.6-h) ATCC25923 Staphylococcusepidermidis 14.5 27.6 (13.1-h)  28.0 (13.5-h) ATCC12228 Streptococcuspyogenes 15.2 32.9 (17.7-h)  40.0 (24.8-h) ATCC19615 Candida albicans8.1 >48.0 (>40-h)   >48.0 (>40-h) 

Antioxidant Activity of ANGex

Ferric reducing/antioxidant power (FRAP) assay (Benzie I F, et al.,Methods in Enzymology: Oxidants and Antioxidants, ed. L Packer, pp15-27, Orlando: Academic Press, 1999) with minor modifications has beenused to measure the antioxidant activity of ANGex complexes. The FRAPreagent was prepared by mixing 40 mL of 0.3 M acetate buffer (pH 3.6), 4mL of 20 mM ferric chloride, and 4 mL of 10 mM TPTZ[2,4,6-Tris(2-pyridyl)-s-triazine]. Serial dilutions (0.1 to 1.0 mM) of6-OH-2,5,7,8-tetramethyl chroman-2-carboxylic acid (CAS 53188-07-1) wereused as FRAP standards. All reagents were brought to 37° C. prior to theassay. FRAP assay was performed in a 96-well microplate by mixing 20 μLof DI water, 10 μL of ANGex complex sample, and 150 μL of FRAP reagent.In combination studies 10 μL of DI water and 20 □1 of ANGex were mixedwith 150 μL of FRAP reagent. After instant incubation at 37° C. for 5min (for ascorbic acid) and for a time lapse of 5 min to 24 h (for ANGexand milk-derived ANG) the absorbance of reaction mixtures was measuredat 593 nm (Spectramax 340PC).

The FRAP reaction kinetics (measured as the rate of increase inabsorbance of reaction mixtures at 593 nm) of ANGex was compared withits source material, the (fdn)-ANG from bovine milk, (fdn)-LF frombovine milk and antioxidant standards (i.e. vitamin-C, TROLOX andFeSO4).

FIGS. 5 A & B shows the antioxidant activity of ANGex as determined bykinetic FRAP assay by measuring the increase in absorbance at 593 nm.The antioxidant efficiency (r) was measured as change in absorbance withthe concentration of ANGex formed at different compositions. In FIG. 5A,the FRAP activity of ANGex formed by the interaction of 694 μM of(fdn)-ANG with 125 μM of (fdn)-LF, is compared with the correspondingindividual activities of the same concentration of (fdn)-LF and(fdn)-ANG, under the same test conditions. All the systems studied showactivity in three stages, with a gradual rise in the initial 14 hours, asteep rise in the period 14-18 hours and saturation in the final 6 oftwenty four hours. ANGex showed consistently higher absorbancethroughout the duration of the experiment. After the initial 14-h, ANGexhad an absorbance of 0.47, while (fdn)-forms of ANG and LF were at 0.29and 0.14, respectively. After 24-h, the maximum absorbance at 593 nm,attained by (fdn)-LF and (fdn)-ANG are 0.87 and 0.64, respectively,whereas ANGex demonstrated a maximum absorbance of 1.18, which is a 35%increase from that of (fdn)-LF and a 84% increase from that of(fdn)-ANG. FIG. 5B shows that when individually tested, (fdn)-ANGdemonstrated antioxidant efficiency (r) of 0.65 Abs/mM. When complexedwith 25 μM of LF, the ‘r’ value increased to 0.67, indicating anenhancement of the antioxidant power. At the same, ANG complexed withhigher concentrations, 75 and 125 μM of LF showed significantly reduced‘r’ values of 0.3 and 0.38, respectively.

DEFINITION OF TERMS

Angiogenic activity is the chemical stimulation of hemovasculardevelopment in tissue. It is generally associated with diffusiblesubstances produced by a variety of cell types.

Angiogenin: As used herein, “angiogenin” or “ANG” refers to anangiogenic-stimulating factor, which is also 14-kDa heparin-bindingprotein that occurs in most cells, also present in various biologicalfluids such as plasma and milk.

ANGex: Refers to immobilized or conjugated angiogenin complexes withnaturally occurring substrates.

Angiogenic stimulators: As used herein, “angiogenic stimulators” refersto growth factors that stimulate neogenesis and regeneration of vasculartissue including the cardiac muscles. These growth factors include, butnot limited to, angiogenin, vascular endothelial growth factor (VEGF),fibroblast growth factor-acidic (FGF-a), fibroblast growth factor-basic(FGF-b), tumor necrosis factor (TNF)-a, and transforming growth factor(TGF)-α/β.

Angiogenic inhibitors: As used herein, “angiogenic inhibitors” refers tophysiological regulators that inhibit biosynthesis of vascularizationprocess. These regulatory compounds include, but not limited toangiostatin, thrombospondin-1 and interferon-α/β, platelet factorfactor-4, 16 kDa N-terminal fragments of prolactin, and endostatin.

Galactose-rich polysaccharide (GRP): Suitable galactose-richpolysaccharides include galactose derivatives comprising galactose,anhydrogalactose, 2-O-methyl-galactose, and 4-O-methyl-galactose, amongothers. The GRP substrates can be purchased or extracted from commercialagars by known methods. Acid hydrolysis of agar under mild conditionsleads to cleavage of glycosidic bonds between C-l of3,6-anhydro-L-galactose and O-3 of D-galactose residues that exposesD-galactosyl residues and leads to the formation of galactose-richpolysaccharides (GRP) (Gray G. Arch. Biochem. Biophys. 163:426-428,1974; Lee R T, et al., Biochemistry 19:156-163, 1980).

Lactoferrin (LF): As used herein, “lactoferrin”, or “LF” refers tovarious protein preparations and forms, including but not limited to,lactoferrin-(tcr) (as described in Naidu U.S. Pat. No. 7,125,983),freely-dispersed native (fdn)-lactoferrin which includes metal-saturated(holo), partially saturated and metal-free (apo) forms of LF. TheLF-bound metal is preferably copper, and other bound metals includezinc, iron, manganese, chromium, aluminum and gallium. The term LFfurther refers to fully and partially glycosylated polypeptide chains ofLF, incomplete polypeptide chains including half-molecules comprising C-and N-terminus lobes of LF. The term LF categorically excludesaggregated-LF and immobilized (Im)-LF forms (as described in Naidu U.S.Pat. No. 6,172,040 B1, issued Jan. 9, 2001) that are devoid of any(fdn)-LF.

Mitogenic activity is the chemical stimulation of cell division.

Ribonuclease (RNAse) activity is characterized by the degradation oflarge RNA molecules, such as the 28S and 18S ribosomal RNAs, to lowermolecular weight species.

Freely-dispersed native (fdn): As used herein, “freely-dispersed native”(fdn) refers to isolated protein molecules free of auto-aggregation orpolymerization and free from binding or immobilization to othersubstrates.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. Therefore, it should be clearly understood thatthe forms of the present invention are illustrative only and are notintended to limit the scope of the present invention.

What is claimed is:
 1. An antimicrobial composition comprisingosteopontin, an isolated angiogenin non-covalently complexed tolactoferrin and at least one antimicrobial protein selected from thegroup consisting of lactoperoxidase (LPO), myeloperoxidase (MPO),salivary peroxidase (SPO) and lysozyme.
 2. The antimicrobial compositionof claim 1, wherein the angiogenin is isolated from a biological fluid.3. The antimicrobial composition of claim 2, wherein the biologicalfluid is selected from the group consisting of colostrum, milk, whey,milk serum, blood, plasma and serum.
 4. The antimicrobial composition ofclaim 2, wherein the biological fluid is obtained from a mammal andwherein the mammal is selected from the group consisting of cows,buffalos, horses, sheep, pigs and camels.
 5. The antimicrobialcomposition of claim 4, wherein the mammal is genetically modified. 6.The antimicrobial composition of claim 1, further comprising nativeangiogenin.
 7. The antimicrobial composition of claim 6, wherein thecomposition comprises complexed angiogenin and native angiogenin at aratio of 1:1 to 1:10.
 8. The antimicrobial composition of claim 6, whichfurther comprises a buffer system, a physiological acceptable base and asalt.
 9. The antimicrobial composition of claim 8, wherein the buffersystem is selected from the group consisting of oxalic acid,ethylenediamine tetraacetic acid and citric acid.
 10. The antimicrobialcomposition of claim 8, wherein the physiological acceptable base issodium bicarbonate.
 11. The antimicrobial composition of claim 8,wherein the salt is selected from the group consisting of sodiumchloride, potassium chloride and calcium chloride.
 12. A method ofinhibiting microbial growth comprising administering the composition ofclaim 1 to a subject in need thereof.
 13. The method of claim 12,wherein the microbe is selected from the group consisting of Escherichiacoli, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiellapneumonia, Staphylococcus aureus, Staphylococcus epidermidis,Streptococcus pyogenes, and Candida albicans.